RESUMO
The present study was to investigate the inhibitory effect and underlying mechanism of Tormentic acid (TA) on hepatic stellate cells (HSCs). HSC-T6 cells were stimulated with Platelet-derived growth factor-BB (PDGF-BB) and TA, and then cell proliferation, apoptosis, inflammatory factor, and collagen-related indicators were detected. In order to elucidate the potential mechanism, the PI3K/Akt/mTOR and NF-κB signalling pathways were also detected. The results showed that TA treatment markedly inhibited PDGF-BB-stimulated HSC-T6 cell activation, as evidenced by the inhibition of cell proliferation, migration and colony formation, as well as the decreased expression of TGF-ß and α-SMA. TA treatment caused a significant increase in the activity of lactate dehydrogenase and significantly promoted cell apoptosis. TA treatment significantly reduced aspartate aminotransferase, alanine aminotransferase and total bilirubin activity. Importantly, TA inhibited the expression of collagen type I and III, alleviating the excessive deposition of extracellular matrix (ECM). Further experiments showed that TA administration significantly inhibited the phosphorylation of PI3K, Akt, FAK and mTOR and the protein expression of P70S6K, indicating the inhibition of the PI3K/Akt/mTOR pathway. Moreover, treatment with TA markedly decreased the phosphorylation of IκBα, NF-κB p65 and IKKα/ß, thereby blocking the NF-κB signal transduction. In summary, this study demonstrates that TA significantly inhibits HSC activation and promotes cell apoptosis via the inhibition of the PI3K/Akt/mTOR and NF-κB signalling pathways. SIGNIFICANCE OF THE STUDY: Tormentic acid (TA) could inhibit HSC activation and alleviate collagen-based ECM deposition, suggesting that TA exerted anti-hepatic fibrosis. Further mechanism research revealed that the inhibition of TA on HSC activation might be through blocking the PI3K/Akt/mTOR and NF-κB signalling pathways. These findings provided a new cue to understand the protective effect of TA against liver fibrosis, which may provide a potential nature medicine for the treatment of liver fibrosis.
Assuntos
Células Estreladas do Fígado/metabolismo , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Triterpenos/farmacologia , Linhagem Celular , Células Estreladas do Fígado/citologia , HumanosRESUMO
The purpose of this study was to investigate the effect of Raf kinase inhibitor protein (RKIP) on the growth, apoptosis, invasion, and metastasis of human hepatic stellate cell line (LX-2). A recombinant plasmid (pcDNA3.1-RKIP) or RKIP-targeting small interfering RNA (siRNA) vector (siRNA-RKIP) was transfected into LX-2 cells to interfere with the RKIP expression. The results demonstrated that increased RKIP expression significantly reduced cell viability, clonogenic growth, and invasion. Further, it promoted cell apoptosis and induced cell cycle arrest in the G1 phase. Overexpression of RKIP led to inactivation of LX-2 cells, as evidenced by the decrease in the expression levels of collagen I and α-smooth muscle actin (α-SMA). In addition, increased RKIP expression significantly reduced the phosphorylation of Raf/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), the transcriptional activity of nuclear factor-κB (NF-κB), and the levels of matrix metalloproteinases-1 and -2. In conclusion, these findings clearly demonstrate that RKIP inhibits LX-2 cell growth, metastasis, and activation, primarily by downregulating the ERK/MAPK and NF-κB signaling pathways.
Assuntos
Células Estreladas do Fígado/citologia , Cirrose Hepática/genética , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Actinas/genética , Actinas/metabolismo , Linhagem Celular , Movimento Celular , Proliferação de Células , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/metabolismo , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Fosforilação , Plasmídeos/genética , RNA Interferente Pequeno/farmacologia , Transdução de SinaisRESUMO
The present study was to investigate the inhibitory effect of methyl helicterate (MH) on hepatic stellate cells (HSC-T6), primarily elucidating the underlying mechanism of MH against liver fibrosis. HSC-T6 cells were activated by platelet-derived growth factor (PDGF) stimulation, and then the effects of MH on cell viability, cytomembrane integrity, colony, migration, apoptosis, and cell cycle were detected. Moreover, the regulative mechanism of MH on HSCs was investigated by detecting the activation of the extracellular signal-regulated kinase (ERK1/2) signaling pathway. The results showed that MH significantly inhibited HSC-T6 cell viability and proliferation in a concentration-dependent manner. It notably promoted the release of lactate dehydrogenase, destroying cell membrane integrity. MH also markedly inhibited HSC-T6 cell clonogenicity and migration. Moreover, MH treatment significantly induced cell apoptosis and arrested cell cycle at the G2 phase. The further study showed that MH inhibited the expression of ERK1, ERK2, c-fos, c-myc, and Ets-1, blocking the ERK1/2 pathway. In conclusion, this study demonstrates that MH significantly inhibits HSC activation and promotes cell apoptosis via downregulation of the ERK1/2 signaling pathway.
Assuntos
Apoptose/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/tratamento farmacológico , Triterpenos/farmacologia , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Matriz Extracelular/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , L-Lactato Desidrogenase/metabolismo , Cirrose Hepática/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , RatosRESUMO
Glycosylation changes associated with cellular transformation can facilitate the growth and progression of tumors. Previously we discovered that the gene Mgat3 encoding the glycosyltransferase GnT-III is elevated in epithelial ovarian carcinomas (EOCs) and leads to the production of abnormal truncated N-linked glycan structures instead of the typical bisected forms. In this study, we are interested in discovering how these abnormal glycans impact the growth and progression of ovarian cancer. We have discovered using stable shRNA gene suppression that GnT-III expression controls the expansion of side-population cells, also known as cancer stem cells. More specifically, we found that GnT-III expression regulates the levels and activation of the heavily glycosylated Notch receptor involved in normal and malignant development. Suppression of GnT-III in EOC cell lines and primary tumor-derived cells resulted in an inhibition of Notch signaling that was more potent than pharmacologic blockage of Notch activation via γ-secretase inhibition. The inhibition resulted from the redirection of the Notch receptor to the lysosome, a novel mechanism. These findings demonstrate a new role for bisecting glycosylation in the control of Notch transport and demonstrate the therapeutic potential of inhibiting GnT-III as a treatment for controlling EOC growth and recurrence.
Assuntos
Carcinoma/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Ovarianas/metabolismo , Receptores Notch/agonistas , Transdução de Sinais , Animais , Carcinoma/patologia , Carcinoma/terapia , Linhagem Celular Tumoral , Feminino , Glicosilação , Humanos , Estimativa de Kaplan-Meier , Camundongos Endogâmicos NOD , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , Invasividade Neoplásica , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/terapia , Ovário/metabolismo , Ovário/patologia , Processamento de Proteína Pós-Traducional , Interferência de RNA , Terapêutica com RNAi , Receptores Notch/metabolismo , Bancos de Tecidos , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND/AIMS: Previous studies have shown that trolline possesses various forms of pharmacological activity, including antibacterial and antiviral potency. The present paper addressed the putative hepatoprotective effects of trolline. METHODS: Rats received 2 ml/kg CCl4 (mixed 1: 1 in peanut oil) intragastrically twice a week for 8 weeks to induce hepatic fibrosis. The animals were then treated with trolline for additional 4 weeks. Liver pathology and collagen accumulation were observed by hematoxylin-eosin and Masson's trichrome staining, respectively. Serum transaminase activity and collagen-related indicator level were determined by commercially available kits. NF-κB pathway activation was also examined. Moreover, the effects of trolline on hepatic stellate cell (HSC-T6) apoptosis, mitochondrial membrane potential (MMP), and autophagy were assessed. RESULTS: Trolline significantly alleviated CCl4-induced liver injury and notably reduced the accumulation of collagen in liver tissues. Trolline treatment also markedly decreased inflammatory cytokines levels by inhibiting the NF-κB pathway. Trolline strongly inhibited HSC-T6 activation and notably induced cell apoptosis by modulating the Bax/Bcl-2 ratio, caspase activity, and MMP. Moreover, trolline significantly inhibited HSC-T6 autophagy, as evidenced by the decrease in the formation of autophagic vacuoles and the number of autophagosomes, by regulating the expression levles of LC3, Beclin-1, P62, Atg 5 and 7. CONCLUSION: Our study demonstrates that trolline ameliorates liver fibrosis, possibly by inhibiting the NF-κB pathway, promoting HSCs apoptosis and suppressing autophagy.
Assuntos
Alcaloides/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Beclina-1/metabolismo , Tetracloreto de Carbono/toxicidade , Linhagem Celular , Colágeno/metabolismo , Citocinas/metabolismo , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/patologia , Cirrose Hepática/prevenção & controle , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , NF-kappa B/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND: Didymin has been reported to have anti-cancer potential. However, the effect of didymin on liver fibrosis remains illdefined. METHODS: Hepatic fibrosis was induced by CCl4 in rats. The effects of didymin on liver pathology and collagen accumulation were observed by hematoxylin-eosin and Masson's trichrome staining, respectively. Serum transaminases activities and collagen-related indicators levels were determined by commercially available kits. Moreover, the effects of didymin on hepatic stellate cell apoptosis and cell cycle were analyzed by flow cytometry. Mitochondrial membrane potential was detected by using rhodamine-123 dye. The expression of Raf kinase inhibitor protein (RKIP) and the phosphorylation of the ERK/MAPK and PI3K/Akt pathways were assessed by Western blot. RESULTS: Didymin significantly ameliorated chronic liver injury and collagen deposition. It strongly inhibited hepatic stellate cells proliferation, induced apoptosis and caused cell cycle arrest in G2/M phase. Moreover, didymin notably attenuated mitochondrial membrane potential, accompanied by release of cytochrome C. Didymin significantly inhibited the ERK/MAPK and PI3K/Akt pathways. The effects of didymin on the collagen accumulation in rats and on the biological behaviors of hepatic stellate cells were largely abolished by the specific RKIP inhibitor locostatin. CONCLUSION: Didymin alleviates hepatic fibrosis by inhibiting ERK/MAPK and PI3K/Akt pathways via regulation of RKIP expression.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/uso terapêutico , Glicosídeos/uso terapêutico , Cirrose Hepática/tratamento farmacológico , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Alanina Transaminase/sangue , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/sangue , Caspases/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Citocromos c/metabolismo , Flavonoides/farmacologia , Glicosídeos/farmacologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática/sangue , Cirrose Hepática/enzimologia , Cirrose Hepática/patologia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND/AIMS: Raf kinase inhibitory protein (RKIP) is closely associated with numerous tumors and participates in their development through regulating the growth, apoptosis, invasion and metastasis of tumor cells. However, the role of RKIP in chronic liver injury and particularly in liver fibrosis is still unclear. METHODS: In the present study, hepatic fibrosis was induced by porcine serum (PS) in rats and primary hepatic stellate cells (HSCs) were isolated from rat livers. Moreover, locostatin was used to interfere with RKIP expression. RESULTS: RKIP expression was significantly inhibited by locostatin in both liver tissues of rats and primary HSCs. Down-regulating RKIP expression resulted in serious liver injury, extensive accumulation of collagen, and significant increase in the levels of ALT, AST and TNF-α during liver fibrosis in rats. Moreover, down-regulating RKIP significantly promoted HSCs proliferation and colony formation in vitro. Reduced RKIP significantly increased the production of collagen and the level of α-SMA as well as the expression of MMP-1 and MMP-2 in both liver tissues and primary HSCs. Furthermore, down-regulating RKIP promoted the activation of the ERK and TLR4 signaling pathways. CONCLUSION: Our findings clearly indicate an inverse correlation between RKIP level and the degree of the liver injury and fibrosis. The decrease in RKIP expression may exacerbate chronic liver injury and liver fibrosis.
Assuntos
Progressão da Doença , Regulação para Baixo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Actinas/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Proliferação de Células , Células Cultivadas , Colágeno/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/genética , Masculino , Metaloproteinase 1 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Ratos Sprague-Dawley , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
[This retracts the article DOI: 10.3892/etm.2018.6542.].
RESUMO
Hypoglycosylation and reduced laminin-binding activity of alpha-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated alpha-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact alpha-dystroglycan, and solid-phase assays determined laminin binding levels to be approximately 50% of normal. In contrast, intact alpha-dystroglycan is undetectable in the dystrophic Large(myd) mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact alpha-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of alpha-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of alpha-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of alpha-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.
Assuntos
Distroglicanas/metabolismo , Laminina/metabolismo , Distrofias Musculares/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Animais , Modelos Animais de Doenças , Técnicas de Introdução de Genes , Glicosilação , Humanos , Laminina/genética , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Distrofias Musculares/genética , Distrofias Musculares/terapia , Mutagênese Insercional , N-Acetilglucosaminiltransferases/genética , Ligação Proteica , Proteínas/genética , Proteínas/metabolismo , TransferasesRESUMO
The TMEM165 gene encodes for a multiple pass membrane protein localized in the Golgi that has been linked to congenital disorders of glycosylation. The TMEM165 protein is a putative ion transporter that regulates H+/Ca++/Mn++ homeostasis and pH in the Golgi. Previously, we identified TMEM165 as a potential biomarker for breast carcinoma in a glycoproteomic study using late stage invasive ductal carcinoma tissues with patient- matched adjacent normal tissues. The TMEM165 protein was not detected in non-malignant matched breast tissues and was detected in invasive ductal breast carcinoma tissues by mass spectrometry. Our hypothesis is that the TMEM165 protein confers a growth advantage to breast cancer. In this preliminary study we have investigated the expression of TMEM165 in earlier stage invasive ductal carcinoma and ductal carcinoma in situ cases. We created a CRISPR/Cas9 knockout of TMEM165 in the human invasive breast cancer cell line MDAMB231. Our results indicate that removal of TMEM165 in these cells results in a significant reduction of cell migration, tumor growth, and tumor vascularization in vivo. Furthermore, we find that TMEM165 expression alters the glycosylation of breast cancer cells and these changes promote the invasion and growth of breast cancer by altering the expression levels of key glycoproteins involved in regulation of the epithelial to mesenchymal transition such as E-cadherin. These studies illustrate new potential functions for this Golgi membrane protein in the control of breast cancer growth and invasion.
RESUMO
Tumor-specific glycosylation changes are an attractive target for the development of diagnostic and therapeutic applications. Periostin is a glycoprotein with high expression in many tumors of epithelial origin including ovarian cancer. Strategies to target the peptide portion of periostin as a diagnostic or therapeutic biomarker for cancer are limited due to increased expression of periostin in non-cancerous inflammatory conditions. Here, we have screened for antibody fragments that recognize the tumor-specific glycosylation present on glycoforms of periostin containing bisecting N-glycans in ovarian cancer using a yeast-display library of antibody fragments, while subtracting those that bind to the periostin protein with glycoforms found in non-malignant cell types. We generated a biotinylated form of a fully human scFv antibody (scFvC9) that targets the bisecting N-glycans expressed by cancer cells. Validation studies in vitro and in vivo using scFvC9 indicate this antibody can be useful for the development of diagnostic, imaging, and therapeutic applications for cancers that express the antigen.
Assuntos
Anticorpos de Cadeia Única/metabolismo , Animais , Western Blotting , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Feminino , Glicosilação , Humanos , Imunoquímica , Fragmentos de Imunoglobulinas/metabolismo , Imageamento por Ressonância Magnética , Camundongos , Neoplasias Ovarianas/metabolismo , Biblioteca de Peptídeos , Polissacarídeos/metabolismoRESUMO
Acute liver injury is a life-threatening syndrome that often caused by hepatocyte damage and is characterized by inï¬ammatory and oxidative responses. Helenalin isolated from Centipeda minima (HCM) has been found to have anti-inflammatory and anti-oxidative effects. Here, this study aimed to investigate the effects and underlying mechanisms of HCM on Lipopolysaccharide/D-Galactosamine (LPS/D-GalN)-induced acute liver injury. Mice were intragastrically administered with various dose of HCM for 10 days; 2â¯h after the final treatment, the mice were injected with 50⯵g/kg LPS and 800â¯mg/kg D-GalN. The histopathological changes, hepatocyte apoptosis, serum cytokines, oxidative stress and inflammatory cytokines were assessed. The results showed that HCM signiï¬cantly ameliorated the hepatic injury, as evidenced by the attenuation of histopathological changes and the decrease in serum aminotransferase and total bilirubin activities. HCM markedly decreased hepatocyte apoptosis by modulating the mitochondria-dependent pathway, including the increase in the Bcl-2/Bax ratio, the inhibition of caspase-3, -8 and -9, and the inhibition of cytochrome C release. Moreover, HCM strongly alleviated oxidative stress, lipid peroxidation and reactive oxygen species (ROS) generation by activating the Nrf2 signaling pathway. In addition, HCM signiï¬cantly attenuated inflammatory cytokines including TNF-α, IL6 and IL-1ß as well as NO production by inhibiting TLR4 signaling transduction and NF-κB activation. In conclusion, HCM protects hepatocytes from damage induced by LPS/D-GalN, which may contribute to its ability to alleviate hepatocyte apoptosis by protecting the mitochondrial function, inhibit oxidative stress by activating the Nrf2 pathway, and attenuate inflammation by inhibiting NF-κB activation. This study demonstrates that HCM may be developed as a potential agent for the treatment of acute liver failure.
Assuntos
Fígado/lesões , Mitocôndrias/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Substâncias Protetoras/uso terapêutico , Sesquiterpenos de Guaiano/uso terapêutico , Transdução de Sinais , Doença Aguda , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Galactosamina , Heme Oxigenase-1/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/patologia , Mediadores da Inflamação/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipopolissacarídeos , Fígado/efeitos dos fármacos , Fígado/patologia , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Óxido Nítrico/metabolismo , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Sesquiterpenos de Guaiano/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Methyl helicterate (MH) has been reported to have protective effects against CCl4-induced hepatic injury and fibrosis in rats, but its protective mechanism, especially on hepatic stallete cells (HSCs), remains unclear. Recently, our pilot experiment showed that MH could inhibit miR-21 expression in HSC-T6 cells, suggesting that miR-21 may be one of the targets of MH to intervene liver fibrosis. To verify the hypothesis, the present study would focus on the regulatory effect of MH on the miR-21-mediated ERK and TGF-ß1/Smads pathways. Briefly, rats were intraperitoneally injected with 0.5â¯ml porcine serum (PS) twice a week for 24â¯weeks to induce liver fibrosis, and meanwhile, the rats were treated with MH from weeks 16 to 24. In vitro experiment, miR-21 expression in HSC-T6 cells was up- or down-regulated using lentiviral transfection assay. Collagen accumulation, inflammatory cytokines, cell apoptosis, miR-21 expression, and activation of the ERK and TGF-ß1/smad2/3 pathways were then assessed. The results showed that MH treatment markedly alleviated PS-induced liver injury, as evidenced by the attenuation of histopathological changes and the decrease in serum alanine and aspartate aminotransferases activity. MH significantly decreased the content of inflammatory cytokines and recruited the anti-oxidative defense system. Moreover, MH treatment significantly decreased miR-21 expression and inhibited the activation of the ERK and TGF-ß1/smad2/3 pathways in liver tissues. In vitro experiments showed that MH strongly inhibited HSC-T6 cell activation and reduced collagen accumulation. Interestingly, miR-21 overexpression significantly promoted HSC-T6 cell proliferation, reduced HSC apoptosis, and increased collagenation, while these abnormal changes induced by miR-21overexpression were significantly reversed by MH treatment. Furthermore, miR-21 overexpression notably activated the ERK and TGF-ß1/Smads pathways via repressing SPRY2 and Smad7 expression respectively, however, these effects were largely abolished by MH treatment. In conclusion, our study demonstrates that MH significantly alleviates PS-induced liver injury and fibrosis by inhibiting miR-21-mediated ERK and TGF-ß1/Smads pathways.
Assuntos
Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/induzido quimicamente , Fígado/patologia , MicroRNAs/genética , Triterpenos/uso terapêutico , Animais , Apoptose , Linhagem Celular , Proliferação de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Células Estreladas do Fígado/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Ratos , Ratos Wistar , Soro , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Suínos , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Accumulating evidence has indicated that Raf kinase inhibitor protein (RKIP) is involved in several intracellular signaling pathways; its abnormal expression is associated with tumor progression and metastasis in several human neoplasms. However, the role of RKIP in acute liver injury has remained elusive. In the present study, acute liver failure was induced by thioacetamide in mice, and locostatin was used to interfere with RKIP expression. It was found that RKIP expression was significantly inhibited by locostatin. Down-regulation of RKIP expression resulted in severe liver injury and extensive release of alanine aminotransferase and aspartate aminotransferase. In addition, reduced RKIP expression significantly enhanced the levels of reactive oxygen species and the content of pro-inflammatory factors such as tumor necrosis factor-α as well as interleukin-6 and -1ß, and decreased the levels of nuclear factor E2-related factor-2 and heme oxygenase-1. Furthermore, down-regulation of RKIP promoted the activation of the nuclear factor-κB and extracellular signal-regulated kinase signaling pathways. In conclusion, the present study indicates an inverse correlation between RKIP level and the degree of hepatic injury, that is, a decrease in RKIP expression may exacerbate acute liver failure.
RESUMO
The present study was to investigate the inhibitory effect of Gypsophila elegans isoorientin-2â³-O-α-l-arabinopyranosyl (GEI) on hepatic stellate cells (HSCs), to reveal the underlying mechanism of GEI against hepatic fibrosis. Our study showed that GEI significantly alleviated liver injury induced by porcine serum (PS) in rats; it notably alleviated collagen accumulation as evidenced by a significant decrease in the levels of collagen biomarkers including hyaluronic acid, laminin, hydroxyproline and procollagen III N-terminal peptide. Moreover, GEI treatment markedly decreased the secretion of inflammatory cytokines by inhibiting the NF-κB pathway and significantly inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and tissue inhibitor of matrix metalloproteinases (TIMPs). Additionally, the cell experiments in vitro showed that GEI strongly inhibited HSC proliferation, migration and clonogenicity and markedly induced HSC apoptosis. Moreover, GEI caused cell cycle arrest at G2 phase. In conclusion, our study demonstrates that GEI significantly alleviates PS-induced hepatic fibrosis by inhibiting the NF-κB pathway, restoring the balance between MMPs and TIMPs, and suppressing HSC activation.
Assuntos
Anti-Inflamatórios/uso terapêutico , Dissacarídeos/uso terapêutico , Flavonas/uso terapêutico , Células Estreladas do Fígado/efeitos dos fármacos , Hepatopatias/tratamento farmacológico , Fígado/patologia , Animais , Apoptose/efeitos dos fármacos , Caryophyllaceae/imunologia , Modelos Animais de Doenças , Fibrose , Humanos , Masculino , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Soro/imunologia , Transdução de Sinais , SuínosRESUMO
In the present study, a flavonoid was isolated from Origanum vulgare and identified as didymin. The effect and mechanism of O. vulgare didymin (OVD) on human HepG2 liver carcinoma cell was then assessed. Our results showed that OVD strongly inhibited the viability, clonogenicity and migration of HepG2 cells. OVD significantly induced apoptosis and induced cell cycle arrest at G2/M phase by regulating cyclin B1, cyclin D1 and CDK4. The anti-proliferative and pro-apoptotic effects were associated with changes in the Bcl-2/Bax ratio and induction of caspase-mediated apoptosis. Moreover, OVD attenuated the mitochondrial membrane potential, accompanied by the release of cytochrome c. In addition, OVD inhibited the ERK/MAPK and PI3K/Akt pathways by increasing the level of Raf kinase inhibitor protein (RKIP). Our study indicates that OVD induces apoptosis against of HepG2 cells through mitochondrial dysfunction and inactivation of the ERK/MAPK and PI3K/Akt pathways by up-regulating RKIP.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Flavonoides/farmacologia , Glicosídeos/farmacologia , Neoplasias Hepáticas/patologia , Mitocôndrias/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Regulação para Cima/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Clonais , Citocromos c/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Gypsophila elegans has been used as a traditional herbal medicine for treating immune disorders and chronic liver diseases in China. The aim of this study is to isolate an active ingredient from this herb and investigate its anti-tumor activity. MATERIALS AND METHODS: An active ingredient was isolated from the ethanol extract using bioassay-guided screening. And its anti-tumor activity was analyzed by testing the cytotoxicity, lactate dehydrogenase (LDH) release, clonogenecity and migration in HepG2 cells. To investigate its potential mechanism, cell apoptosis, cell cycle arrest, reactive oxygen species (ROS), cytochrome c, mitochondria membrane potential (MMP) and caspase level were determined in liver cancer cell line HepG2. RESULTS: A flavonoid glycoside, i.e., G. elegans isoorientin (GEI), was isolated from this herb and identified as Isoorientin-2â³-O-α-l-arabinopyranosyl. Our results showed that GEI significantly inhibited the viability and proliferation of HepG2 cells in a dose- and time-dependent manner, and its cytotoxic effect was also confirmed by the elevated level of LDH. GEI treatment could markedly inhibit the clonogenicity and migration of HepG2 cells. Moreover, GEI induced remarkable apoptotic death of HepG2 cells through cell cycle arrest at G1 phase via the regulation of cell cycle-related genes, such as cyclin D, cyclin E and CDK2. Further study showed that GEI treatment significantly elevated ROS formation, followed by attenuation of MMP via up-regulation of Bax and down-regulation of Bcl-2, accompanied by cytochrome c release to the cytosol. In addition, GEI treatment resulted in a significant dose-dependent increase in caspase-3 and -9 proteolytic activities. CONCLUSION: The present study demonstrates that the ability of GEI to induce apoptosis against HepG2 cells mediated by mitochondrial-mediated pathway.